Project summary Filoviruses are among the deadliest known human viruses. The family Filoviridae contains two clinically relevant genera; Ebolavirus and Marburgvirus that each consist of multiple viruses. Filoviruses have caused 48 outbreaks since Marburg virus was discovered in 1967. Marburgvirus has caused 13 outbreaks, 5 of which occurred in the past 10 years with an average lethality of 81%. The recent outbreak of Ebola virus disease in West Africa was a reminder of the threat posed by filoviruses to global human health and the impact of filovirus outbreaks on local and global economies. While several monoclonal antibodies (mAbs) and vaccines are in development for ebolaviruses, there is a severe shortage of countermeasures for marburgviruses. Only a single marburgvirus mAb has shown efficacy in nonhuman primates (NHPs) and it requires extremely high doses. Furthermore, relying on a single mAb would leave us vulnerable to potential escape variants. Therefore, there is an urgent need to discover and develop effective broadly neutralizing mAbs (bNAbs) for marburgviruses. Based on our team?s extensive experience with filovirus mAbs, we hypothesize that an effective immunotherapy for marburgvirus should target the receptor binding site and the base of the glycoprotein (GP) trimer in form of a mAb cocktail. To discover such mAbs we have assembled a team of experts with proven track record in mAb discovery, filovirus biology, and animal models. The PI has been on the forefront of developing bNAbs for ebolaviruses using B cells from immunized macaques. Our filovirus expertise is complemented by Dr. Brandon DeKosky who has developed an unparalleled methodology for large-scale interrogation of functional B cell repertoire and high throughput cloning and display of natively paired VH:VL sequences. This unique system will allow rapid isolation of antibodies with desired functional properties. Our team further incorporates Dr. Tom Geisbert of UTMB, one of the most recognized filovirus experts. We have already immunized NHPs by priming with an engineered pseudotype virus and boosting with high quality GP trimers that are engineered to expose critical epitopes. The neutralizing serum antibody response has been characterized, indicating the presence of B cells producing neutralizing antibodies. Immunized macaque B cells from peripheral blood, bone marrow, lymph nodes and spleen are now available for the comprehensive discovery effort outlined in this proposal. We have three specific Aims: In Aim 1, we will apply high throughput antibody screening to mine memory B cells and plasmablasts and isolate natively paired VH:VL clones reactive to Marburgvirus GP. Antibodies identified in Aim 1 will be produced and fully characterized for functionality and mechanism of action in Aim 2. Finally, in Aim 3, we will test up to 20 of the most promising antibodies in a surrogate BSL-2 model in AG129 mice, followed by evaluation of the best lead candidates in the guinea pig model of marburgvirus infection in BSL-4. Upon successful completion of the Phase I we anticipate several advanced candidates for testing in NHPs in a follow- on Phase SBIR II to deliver an effective immunotherapeutic mAb or mAb cocktail for clinical development.